NextSeq 500 sequencing; RNA-seq of wild-type and PGR (progesterone receptor) knockout pluripotent mouse EpiSCs (epiblast stem cells) and differentiated samples derived from these cell lines (primitive streak and mesoderm)
RNA-seq of wild-type and PGR (progesterone receptor) knockout pluripotent mouse EpiSCs (epiblast stem cells) and differentiated samples derived from these cell lines (primitive streak and mesoderm)
Cells were seeded on fibronectin-coated 6-well plates in N2B27 medium (DMEM-F12:Neurobasal, 1:1, N2, B27) supplemented with Activin A, FGF and XAV-939 and differentiated to all desired cell types as described in the publication. At 24h (EpiSC, PPS and APS) and 48h (mesoderm) cells were washed briefly and incubated with accutase for 3 min at 37C. After that time accutase was diluted with 3x volume N2B27, samples were centrifuged and the cell pellet was resuspended in lysis buffer (QIAGEN RNAeasy kit). RNA was extracted using QIAGEN RNAeasy kit according to manufacturer's indications. RNA concentration was quantified by NanoDrop and quality was verified with a Fragment analyzer. Library preparation was carried out using 0.5 μg of RNA with NEBNext Ultra II RNA library Prep Kit (New England BioLabs, E7770S) as per manufacturer's instructions. Libraries were amplified for 4 total PCR cycles and purified with AMPure XP beads. DNA concentration was measured with a Qubit Fluorometer and fragment size was determined with a Fragment analyzer.